Article Characteristics of vesca Yield Parameters and Accumulation under Water Deficit Stress

Rytis Rugienius 1,* , Vidmantas Bendokas 1 , Tadeusas Siksnianas 1, Vidmantas Stanys 1, Audrius Sasnauskas 1 and Vaiva Kazanaviciute 2

1 Lithuanian Research Centre for and Forestry, Department of Orchard Genetics and Biotechnology, Institute of Horticulture, LT-54333 Babtai, Lithuania; [email protected] (V.B.); [email protected] (T.S.); [email protected] (V.S.); [email protected] (A.S.) 2 Department of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius University, LT-10257 Vilnius, Lithuania; [email protected] * Correspondence: [email protected]; Tel.: +370-37-555253

Abstract: Plants exposed to drought stress conditions often increase the synthesis of —natural plant pigments and antioxidants. However, water deficit (WD) often causes significant yield loss. The aim of our study was to evaluate the productivity as well as the anthocyanin content and composition of from cultivated “Rojan” and No. 17 plants (seedlings) grown under WD. The plants were grown in an unheated greenhouse and fully irrigated (control) or irrigated at 50% and 25%. The number of berries per plant and the weight were evaluated every 4 days. The anthocyanin content and composition of berries were evaluated with the same   periodicity using HPLC. The effect of WD on the yield parameters of two evaluated F. vesca genotypes differed depending on the harvest time. The cumulative yield of plants under WD was not less Citation: Rugienius, R.; Bendokas, V.; Siksnianas, T.; Stanys, V.; Sasnauskas, than that of the control plants for 20–24 days after the start of the experiment. Additionally, berries A.; Kazanaviciute, V. Characteristics accumulated 36–56% (1.5–2.3 times, depending on the harvest time) more anthocyanins compared of Fragaria vesca Yield Parameters and with fully irrigated plants. Our data show that slight or moderate WD at a stable air temperature of ◦ Anthocyanin Accumulation under about 20 C positively affected the biosynthesis of anthocyanins and the yield of F. vesca berries. Water Deficit Stress. Plants 2021, 10, 557. https://doi.org/10.3390/ Keywords: anthocyanins; deficit irrigation; drought; pigments; temperature plants10030557

Academic Editors: Alfredo Guéra and Francisco Gasulla Vidal 1. Introduction Abiotic stresses, including drought, have detrimental effects on the yield of vari- Received: 19 February 2021 ous crops in different parts of the world, and agriculture in many regions has become Accepted: 12 March 2021 uncompetitive as a result of this yield instability. Predicted climate changes in the near Published: 16 March 2021 future are expected to cause major challenges in modern agriculture due to their significant potential negative impacts on both the quantity and quality of various crops, including Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in [1,2]. Water deficit is generally associated with a reduction in the size and yield published maps and institutional affil- of strawberry . However, some studies have demonstrated that water deficit might iations. increase the dry matter content, improve the concentration of health-related compounds, and improve the taste of strawberry fruits [3–7]. Declining water resources are leading to concerns about high water usage for some horticultural purposes. Studies to determine the minimum necessary amount of water for crop development are required not only to save water and/or energy, but also to improve plant fitness to resist biotic and abiotic stresses, Copyright: © 2021 by the authors. as well as improve nutritional, functional, and sensorial food properties [5,8]. Licensee MDPI, Basel, Switzerland. This article is an open access article Plants exposed to stress often increase the biosynthesis of phenolic compounds, partic- distributed under the terms and ularly flavonoids, such as anthocyanins. As one of the most ubiquitous classes of flavonoids conditions of the Creative Commons in berries, anthocyanins possess a multitude of biological roles, including protection against Attribution (CC BY) license (https:// solar exposure, and ultraviolet radiation, free radical scavenging and anti-oxidative capac- creativecommons.org/licenses/by/ ity, defense against many different pathogens, attraction of predators for seed dispersal, 4.0/). and the modulation of signaling cascades [9]. Epidemiological and clinical studies suggest

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that an anthocyanin-enriched diet may lower levels of certain oxidative stress biomarkers in humans, and this could be associated with a reduced risk of cognitive decline and the development of neurodegenerative and cardiovascular diseases, in addition to sustaining hepatic function and kidney protecting activities [10]. Berry anthocyanins improve neu- ronal and cognitive brain functions and ocular health as well as protect genomic DNA integrity. Anthocyanins assist in antiplatelet aggregation, act as an antiangiogenic barrier, and display anticancer properties [11,12]. Anthocyanin composition, as well as that of other flavonoids, has been studied exten- sively in wild and cultivated strawberry [13–18].This notwithstanding, the relation between water deficit (WD) and anthocyanin accumulation is not fully understood, especially in crops, and the potential to increase food value and reduce cultivation costs is not fully exploited. Wild strawberry (Fragaria vesca) is a good model plant for various studies because of its short cycle, small , and ease of reproduction [19]. Wild strawberry is a relative of the cultivated strawberry Fragaria × ananassa, which is grown in open fields and greenhouses. The aim of our study was to evaluate the growth and fruiting of F. vesca plants and their anthocyanin content under water deficit in a greenhouse.

2. Results 2.1. F. vesca Yield Parameters The number and yield of berries from fully irrigated control (I100) plants of both “Rojan” and hybrid No. 17 remained relatively stable during the first two weeks of the treatment in April (Figure1a,b,e,f). Later, starting from the fourth–fifth pick, or 20–25 days after the beginning of harvesting, the number and yield of berries started to increase. Berry dynamics were affected by extreme air temperature, which increased to 24 ◦C outside starting on 8 May 2013. The temperature inside the greenhouse sometimes reached 35 ◦C. The rise in temperature accelerated berry ripening and led to increases in the number and yield of ripened berries. This increase was variable and reached a maximum on 20–24 May. The berry size decreased gradually until 8 May. Then, a temporary increase in berry size, especially for hybrid No. 17, was observed (Figure1c,d). This was also clearly a consequence of the increased air temperature. The impact of water deficit on yield parameters was genotype dependent. This impact was much more noticeable in hybrid No. 17 than in “Rojan”. The cumulative berry number of hybrid No. 17 plants grown under water deficit was higher than that of fully irrigated plants for almost the entire treatment period (Figure1b). For 8 days of the experiment, the cumulative yield of 50% irrigated plants (I50) did not differ from that of control (fully irrigated) plants, and their yield subsequently became significantly higher and was the highest at the end of the experiment compared with 25% irrigated (I25) and control plants (Figure1f). The cumulative yield of I25 plants was higher than that of I100 plants until 16 May, which is about two-thirds of the duration of the treatment. During the last 4 days, it became the lowest compared with I50 and control plants. Interestingly, although water deficit significantly (except for 8 May) decreased the average weight of the fruit of this hybrid, for most of the harvesting period, the cumulative yield was higher than that of I100 plants (Figure1d). The cumulative berry number of “Rojan” plants grown under water deficit was also higher than that of control plants for almost the entire duration of the experiment. However, the difference was much smaller than that of No. 17 plants, and at the end of the experiment, the number of berries was the same among all irrigation variants of “Rojan” (Figure1a). The cumulative yield of I50 “Rojan” plants did not differ from that of I100 plants for the entire experimental period. The yield of I25 plants from the 8th to 24th day of the experiment (30 April–20 May) was higher than that of the control, but starting on 24 May, it decreased, and at the end of the experiment, it was the lowest compared with other irrigation variants (Figure1e). The difference in average berry weight among different treatments was also much lower in “Rojan” than in hybrid No. 17 (Figure1c). The berry weight of I50 plants did not differ from that of I100 plants for almost the entire experimental Plants 2020, 9, x FOR PEER REVIEW 3 of 11

Plants 2021, 10, 557 other irrigation variants (Figure 1e). The difference in average berry weight among differ-3 of 11 ent treatments was also much lower in “Rojan” than in hybrid No. 17 (Figure 1c). The berry weight of I50 plants did not differ from that of I100 plants for almost the entire ex- perimental period. Starting on 16 May, the berry weight of I25 plants significantly de- period. Starting on 16 May, the berry weight of I25 plants significantly decreased compared creased compared with that of the control, and it was the lowest at the end of the experi- with that of the control, and it was the lowest at the end of the experiment. This decrease, ment. This decrease, possibly combined with the lack of an increase in the number of ber- possibly combined with the lack of an increase in the number of berries, resulted in I25 ries, resulted in I25 having the lowest yield compared with the control and I50 plants at having the lowest yield compared with the control and I50 plants at the end of experiment. the end of experiment.

FigureFigure 1.1. CumulativeCumulative berry berry number number (a (,ba,),b ),average average berry berry weight weight (c,d (),c ,andd), andcumulative cumulative yield yield per plant per plant(e,f) of (e alpine,f) of alpinestraw- strawberryberry “Rojan” “Rojan” (a,c,e ()a and,c,e) hybrid and hybrid No. 17 No. (b 17,d,f (b) ,ind, fdifferent) in different picks. picks. Plants Plants were were fully fully irrigated irrigated (I100) (I100) and andirrigated irrigated with with half half(I50) (I50) and anda quarter a quarter (I25) (I25) of full of fullirrigation irrigation.. Data Data followed followed by the by thesame same letter letter are arenot notsignificantly significantly different different according according to the to theleast least significant significant difference difference (LSD) (LSD) test test at p at ≤ p0.05.≤ 0.05.

InIn hybridhybrid No.No. 1717 plantsplants underunder halfhalf irrigationirrigation (I50),(I50), thethe totaltotal yieldyield increaseincrease waswas 6.2%6.2% comparedcompared withwith controlcontrol plants.plants. TheThe decreasesdecreases inin totaltotal yieldyield perper plantplant ofof hybridhybrid No.No. 1717 andand “Rojan”“Rojan” I25I25 plantsplants werewere 8.6%8.6% andand 21.0%, 21.0%, respectively. respectively. TheThe experimentexperiment for thethe leastleast irrigatedirrigated (I25)(I25) plantsplants waswas completedcompleted onon 2424 May,May, whenwhen plantsplants becamebecame dry,dry, andand theythey dieddied beforebefore 2828 May.May. ClearClear signssigns ofof wiltingwilting werewere visiblevisible inin thethe middlemiddle ofof May.May.

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2.2. Anthocyanin ProfileProfile andand ConcentrationConcentration inin BerriesBerries duringduring HarvestingHarvesting The anthocyaninanthocyanin concentrationconcentration of of fully fully irrigated irrigatedF. F. vesca vescaberries berries of of both both varieties varieties varied var- fromied from 3.6 to 3.6 4.7 to mg/100 4.7 mg/100 g fresh g fresh weight weight and remainedand remained stable stable until 4until May 4 (Figure May (Figure2a,c). Then, 2a,c). theThen, anthocyanin the anthocyanin concentration concentration decreased decreased more more than two-foldthan two-fold in both in both varieties varieties until until the endthe end of the of harvestingthe harvesting season. season. It was It observedwas observed in our in study our study that WDthat causedWD caused a considerable a consid- increaseerable increase in anthocyanin in anthocyanin content content in wild in strawberry wild strawberry fruits, especiallyfruits, especially at the beginningat the begin- of thening harvest. of the harvest. The berries The berries of “Rojan” of “Rojan I50 and” I50 I25 and plants I25 accumulatedplants accumulated 1.5 and 1.5 2.3 and times 2.3 moretimes anthocyaninsmore anthocyanins then fullythen irrigatedfully irrigated plants plants after after 4 days 4 days of the oftreatment the treatment (Figure (Figure2a). Anthocyanin2a). Anthocyanin concentrations concentrations in the in berries the berries of hybrid of hybrid No. 17 No. were 17 were 1.5–1.6 1.5–1.6 times times higher higher than thosethan those of fully of irrigatedfully irrigated plants plants under under both deficit both deficit irrigation irrigation regimes regimes (Figure 2(Figurec). Differences 2c). Dif- inferences the total in amountthe total of amount berry anthocyanins of berry anthocya obtainednins under obtained full andunder deficit full irrigationand deficit regimes irriga- weretion regimes significant were (p ≤significant0.05) for both(p ≤ 0.05) . for both When cultivars. analyzing When the analyzing cumulative the anthocyanin cumulative contentanthocyanin at the content end of at study, the end we of observed study, we clear observed differences clear betweendifferences irrigation between treatments irrigation startingtreatments on starting the eighth on the day eighth of the day treatment of the treatment for “Rojan” for and“Rojan” on the and 12th on the day 12th for hybridday for No.hybrid 17. No. At the 17. endAt the of theend study, of the thestudy, cumulative the cumulative amount amount of anthocyanins of anthocyanins in berries in berries of I25 bothof I25 “Rojan” both “Rojan” and No. and 17 No. plants 17 plants was 56% was and 56% 36% and higher, 36% higher, respectively, respectively, than thatthan of that fully of irrigatedfully irrigated control control plants plants (Figure (Figure2b,d). 2b,d).

FigureFigure 2.2. DynamicsDynamics ofof totaltotal anthocyaninanthocyanin contentcontent (mg/100g(mg/100g fresh weight)weight) in the berries of wild strawberry “Rojan”“Rojan” ((aa)) andand No.No. 1717 ((cc)) andand cumulative cumulative anthocyanin anthocyanin content content (b (,bd)), and respectively. (d), respectively. Plants were Plants fully were irrigated fully (I100)irrigated and (I100) irrigated and withirrigated half (I50)with andhalf a(I50) quarter and a (I25) quarter of full (I25) irrigation. of full irrigation. Data followed Data fo byllowed the same by the letter same are letter not are significantly not significantly different different according accord to theing to the LSD test at p ≤ 0.05. LSD test at p ≤ 0.05.

We analyzedanalyzed thethe anthocyaninanthocyanin compositioncomposition of F.F. vescavesca berriesberries andand establishedestablished thatthat pelargonidinpelargonidin 3-O-glucoside3-O-glucoside (Pel3G)(Pel3G) waswas thethe mostmost abundantabundant anthocyanin.anthocyanin. InIn “Rojan”“Rojan” andand No. 17 berries,berries, Pel3GPel3G accountedaccounted forfor 50.7%50.7%± ± 7.5% andand 50.6%50.6% ±± 4.4% of all anthocyanins,anthocyanins, respectively,respectively, followed followed by by cyanidin-3-O-glucoside cyanidin-3-O-glucoside (C3G), (C3G), which which accounted accounted for 29.2% for 29.2%± 8.3% ± and8.3% 27.4%and 27.4%± 5.5% ± 5.5% of allof all anthocyanins, anthocyanins, respectively. respectively. The The percentage percentage ofof peonidin-3-O-peonidin-3-O- glucoside waswas 8.4%8.4% ±± 1.2% and 10.1%10.1% ±± 1.7%, respectively. The concentrationsconcentrations ofof otherother unidentifiedunidentified minor anthocyanins were low and togethertogether accountedaccounted for lessless thanthan 10%10% ofof total anthocyanins. In examining the ratio of anthocyanins, we focused on the first two anthocyanins, as their levels were the highest. A tendency of C3G to decrease and Pel3G

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Plants 2020, 9, x FOR PEER REVIEW total anthocyanins. In examining the ratio of anthocyanins, we focused on the first5 of two 11 anthocyanins, as their levels were the highest. A tendency of C3G to decrease and Pel3G to increase duringduring harvestingharvesting waswas clearlyclearly apparent,apparent, although the variationvariation waswas slightslight throughout the experiment. This tendency was more consistent in the berries of “Rojan”. The Pel3G/C3G ratio ratio in in the the berries berries of of this was 1.31 at the start of thethe treatmenttreatment and was relatively stable (up to 12 days after the start of treatment), and then it increased significantly:significantly: on the 28th day, it was 2.9 times higher compared withwith thethe beginningbeginning ofof thethe study period (Figure 33a).a). InIn contrastcontrast toto thisthis variety,variety, aadifferent differenttrend trend was was established establishedin in berries of hybrid No. 17: the Pel3G/C3G ratio ratio decreased decreased at at the the beginning beginning and subsequently began to increase, and it had increased by 1.4 times at the end of the study compared with the start (Figure3 3b).b). TheThe proportionproportion andand dynamicsdynamics ofof bothboth mainmainanthocyanins anthocyanins ininberries berries during harvesting did did not not depend depend on on water water de deficit.ficit. However, However, the the Pel3G/C3G Pel3G/C3G ratio ratio in the in theberries berries of No. of No.17 plants 17 plants irrigated irrigated at 25% at (I25) 25% was (I25) up was to up35% to and 35% 20% and lower 20% in lower the begin- in the beginningning and at and the atend the of end the of study the study compared compared with that with in that fully in fullyirrigated irrigated plants. plants.

Figure 3.3. Pel3G/C3GPel3G/C3G ratio ratio in in the berries of wild strawberry “Rojan” ( a) and No. 17 (b). Plants were fully irrigated (I100) andand irrigatedirrigated with halfhalf (I50)(I50) andand aa quarterquarter (I25)(I25) ofof fullfull irrigation.irrigation. Data followed by the same letter are notnot significantlysignificantly different accordingaccording toto thethe LSDLSD testtest atat pp ≤≤ 0.05.

3. Discussion Water deficitdeficit inin strawberrystrawberry fruits is generallygenerally associated with a reduction in berry size and yield. Previous studies have shown that drought stress decreases yieldyield andand fruitfruit weight, as well as leaf area, leaf dry matter, shoot dry matter, totaltotal dry matter, relativerelative water content, and stomatal conductance [[3,5,20,21].3,5,20,21]. Liu and co-authors [[22]22] showedshowed thatthat water deficitdeficit and partial -zone drying decreaseddecreased the berry weight of thethe strawberrystrawberry (“Honeoye”), butbut thethe berryberry numbernumber remainedremained stable.stable. The impact of our WD treatmentstreatments on differentdifferent yieldyield parametersparameters (berry(berry numbernumber andand berry weight) ofof wildwild strawberrystrawberry depended depended on on the the variety. variety. Insufficient Insufficient irrigation irrigation resulted resulted in decreasedin decreased berry berry weights weights for strawberry for strawberry hybrid hy No.brid 17 No. and, 17 to and, a much to a lesser much extent, lesser “Rojan”, extent, but“Rojan”, it increased but it increased the berry numberthe berry and number yield atan thed yield beginning at the ofbeginning the treatment. of the WDtreatment. stress hasWD beenstress reported has been to reported stimulate to both stimulate berry setboth and berry ripening set and speed ripening [23]. speed Mild WD [23]. was Mild found WD towas increase found appleto increase yield [24]. An yield increase [24]. An in berryincrease yield in perberry plant yield of per a strawberry plant of a hybridstrawberry was observedhybrid was in ourobserved other experimentin our other [25 experiment]. This indicates [25]. This that theindicates dehydration that the stress-induced dehydration increasestress-induced in strawberry increase yield, in strawberry at least during yield, certain at least growth during stages, certain is growth not coincidental. stages, is not coincidental.Differences in berry set in response to water stress between the evaluated varieties mayDifferences be explained in byberry the set genetic in response background to water of hybridstress between No. 17. the One evaluated of its parents varieties is themay woodland be explained strawberry by the geneticF. vesca background, which has probablyof hybrid evolvedNo. 17. One with of different its parents adaptive is the characteristicswoodland strawberry from those F. vesca of the, which cultivated has probably alpine strawberry. evolved with different adaptive char- acteristicsDifferences from those among of genotypesthe cultivated have alpine been observedstrawberry. by other authors investigating the responseDifferences of strawberry among plants genotypes to WD have [20 –been27]. Theobserved reaction by other to water authors deficit investigating stress in plants the ofresponse different of strawberry genotypes hasplants been to WD related [20–27]. to the Th ratioe reaction between to water carbon deficit fixation stress and in plants water lossof different [20] or differencesgenotypes has in the been response related toto jasmonicthe ratio between acid and carbon stomatal fixation closure and speed water [28 loss]. [20] or differences in the response to jasmonic acid and stomatal closure speed [28]. The rise in temperature before 8 May further accelerated the ripening of berries in all studied irrigation regimes. An increase in berry yield due to elevated air temperature was also observed in our other experiment [25]. In both cases, berry weight and yield de- creased at the end of harvest, especially when plants were grown under WD.

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The rise in temperature before 8 May further accelerated the ripening of berries in all studied irrigation regimes. An increase in berry yield due to elevated air temperature was also observed in our other experiment [25]. In both cases, berry weight and yield decreased at the end of harvest, especially when plants were grown under WD. In our opinion, the changes in yield parameters under water deficit conditions in our treatment can be divided into three stages. In the first stage, the air temperature was low and stable, and water deficit caused berry ripening to slightly accelerate. The decrease in the weight of the berries was variable and depended on the variety. In the second stage, when the air temperature and fluctuated, the ripening of the berries accelerated even further, but the stress due to water deficit also increased. The berry size decreased more rapidly under a larger water deficit. In the third stage, the negative effects of water deficit stress, exacerbated by higher temperatures, revealed themselves. The size of the berries decreased substantially, the number of berries did not increase, and the yield decreased. Plants need to endure different abiotic stresses, and polyphenols (including antho- cyanins) accumulate in response to these stresses, helping plants to acclimatize to unfavor- able environments. Hence, the concentration of these metabolites in plant tissue is a good indicator to predict the extent of abiotic stress tolerance in plants, which varies greatly among plant under an array of external factors [29]. As mentioned before, in our experiment, water deficit caused a considerable increase in anthocyanin content in wild strawberry fruits, especially at the beginning of the harvest. According to data from several studies, water stress induces the accumulation of antho- cyanin compounds in the berries of strawberry plants [5,21,30]. Bordonaba and Terry [3] showed that differences exist in the way that different strawberry cultivars respond to drought stress, resulting in different fruit compositions. Despite the detrimental effect that WD can have on berry size, reducing water irrigation by a quarter between flower initiation and fruit harvest has been found to result in better water use efficiency, as well as enhanced fruit quality and taste in some cultivars [3]. Mild drought and salt stress resulted in an increased content of phenolics, anthocyanins, and L-ascorbic acid in strawberry fruits; however, fruit yield was not affected [5]. According to the data from our study, increase in anthocyanin content in wild - berry fruits under water deficit had two peaks: the first was at the beginning of the treatment, and the second was after the middle of the treatment, from 8 May. We propose that the first peak was caused by slight or moderate water deficit stress, and the second peak was the result of air temperature changes, and an increase in sunny hours. It is known that the accumulation of anthocyanins and other polyphenols in plants is influenced by many factors, including light and temperature [29]. Interestingly, anthocyanin accumulation had two peaks in our other experiment [25]. The influence of temperature on anthocyanin biosynthesis is ambiguous according to different investigators. Higher temperature during cultivation generally promotes the synthesis of anthocyanins and other phenolic com- pounds in strawberry fruits [31], but this effect is genotype dependent [1]. According to Wang and Camp [32], a daytime temperature of 30 ◦C inhibited plant and fruit growth and also reduced fruit quality. However, elevated CO2 and a higher temperature enhanced the amounts of accessible bioactive compounds in , including anthocyanins [1]. Generally, temperatures of approximately 25 ◦C favor anthocyanin biosynthesis, whereas higher temperatures, such as 35 ◦C, are associated with anthocyanin degradation [33]. In our treatment, anthocyanin content in the berries of “Rojan” plants grown under water deficit increased at the beginning of harvesting until a sharp temperature increase occurred. However, the increase in temperature and sunlight may have affected the fluctuations in anthocyanin levels in the second half of harvesting. The results of our study show that WD has a positive effect on anthocyanin accumulation in Fragaria berries only at moderate air temperatures of around 20 ◦C. Similar results were obtained in our other study, in which anthocyanin accumulation did not increase under WD at elevated temperatures [25]. In the current experiment, we observed a substantial decrease in the anthocyanin content in berries starting on 13 May, when the air temperature inside the greenhouse increased to Plants 2021, 10, 557 7 of 11

over 30 ◦C. It is probable that anthocyanin synthesis and accumulation reached a certain limit at higher air temperatures, as observed in other studies [34,35]. The anthocyanin content in the berries of hybrid No. 17 decreased 8 days later, on 21 May. This shows that the accumulation of anthocyanins in the berries of hybrid No. 17 is less sensitive to temperature despite the higher amplitude of the change in morphological yield parameters; that is, an increase in berry number and decrease in average berry weight, compared with the “Rojan” variety. These results corroborate the findings of Crespo et al. [36] and Carbone et al. [15], who observed that the strawberry genotype had a dominant role over environmental factors in anthocyanin accumulation. Anthocyanins, phenolic acids, su- crose, and were found to be the most discriminant variables among cultivars, while climatic conditions and the cultivation system were behind changes in polyphenol contents [16]. As mentioned before, 3-O-glucoside (Pel3G) and cyanidin-3-O-glucoside (C3G) in F. vesca berries in our treatment constituted nearly 50.7% and 27.4–29.2% of total anthocyanins, respectively. According to the data of Kawanobu et al. [37], in the berries of wild strawberry, C3G and Pel3G constituted 49.2% and 40.7% of all anthocyanins, respectively. Pel3G clearly predominates, reaching from 50% to more than 90% of total anthocyanin content, while other anthocyanins (Pel3Rut, C3G, and Pel3MG) rarely exceed 30% in F. ananassa, according to data from different studies [13,37,38]. Lower Pel/C3G ratios are thought to be desirable because cyanidin 3-glucoside, in most cases, has greater antioxidant capacity than that of pelargonidin 3-glucoside [39]. Although the content of individual anthocyanins was influenced significantly by the strawberry cultivar, production system, harvest period, climatic conditions, soil, day length, and altitude, the proportions among Pel3G, C3G, and Pel3rut were consistent, suggesting a characteristic anthocyanin distribution [40,41]. In our experiment, an increase in the Pel3G/C3G ratio was observed in the second half of the growing season. This may be related to an increase in air temperature, which started in the middle of the harvest season. The percentages of separate anthocyanins did not differ significantly under different irrigation regimes. The influence of the high temperature, but not the irrigation regime, on the anthocyanin ratio is confirmed by our other experiments [25]. At the beginning of the current treatment, when air temperature was lower, the Pel3G/C3G ratio in the berries of No. 17 grown under the highest water deficit was up to 35% lower than in the berries of normally irrigated plants. It was established that the amount of C3G in berries of the “Rojan” variety was significantly negatively correlated with the number of berries (r = −0.83 ± 0.24) and yield of plants (r = −0.81 ± 0.26) and positively correlated (insignificantly) with the average berry weight (r = 0.56 ± 0.35) (linear correlation). On the contrary, the amount of Pel3G had a positive correlation with the number of berries (r = 0.90 ± 0.19) and a negative correlation with the average berry weight (r = 0.66 ± 0.32). Similar correlations were established in the berries of hybrid No. 17, but the correlation coefficients were lower and often insignificant. It may be concluded that the dynamics of the yield parameters of F. vesca depend on the genotype and temperature changes during berry ripening. Water deficit stress causes an increase in the wild strawberry berry set and yield in the first half of harvesting and a decrease in yield parameters (with the possible exception of the yield of No. 17) at the end. Although WD caused a decrease in the total yield of up to 21.0%, the berries accu- mulated up to 2.3 times more anthocyanins compared with normally irrigated plants. The highest anthocyanin concentration and the lowest pelargonidin 3-O-glucoside/cyanidin-3- O-glucoside ratio (1.1–1.3) were observed in the first part of the fruiting season when the air temperature in the greenhouse was nearly 20 ◦C. The anthocyanin content decreased and the Pel3G/C3G ratio (3.1–3.8) increased in F. vesca berries at elevated air temperatures (over 25 ◦C). The data obtained in this study expands our knowledge about growth changes during harvesting and the accumulation of anthocyanins in F. vesca under WD stress. These insights could help to develop cultivation technology that preserves water resources and Plants 2021, 10, 557 8 of 11

increases the nutritional value of berries during their growth. Our data reveal that for the successful use of reduced irrigation, moderate to severe (25–50% of full irrigation) water deficit treatment at a stable air temperature of approximately 20 ◦C for a maximum of three weeks is desirable for berries with maximal yield and the highest nutritional value. From the obtained data, growers are recommended to reduce watering to 50% at moderate temperatures for at least the first half of the harvest season. This would reduce water consumption and improve the quality of the berries. Data on changes in anthocyanin accumulation under different environmental conditions are important for gene expression studies in order to identify key factors that affect these valuable compounds.

4. Materials and Methods The experiment was conducted at the Lithuanian Research Centre for Agriculture and Forestry, Institute of Horticulture, Babtai Lithuania, located at the coordinates 55◦ 080 N and 23◦ 800 E and an elevation of 55 m. The climate is a humid continental type with an annual average precipitation of 630 mm and an average temperature of −5 ◦C in January and 17.3 ◦C in July. Wild strawberry or European “alpine strawberry” (F. vesca var. semperflorens) “Rojan” (“Rügen”), and hybrid No. 17 [(F. vesca (woodland strawberry)) × F. vesca var. semperflorens]) were used in this research. Strawberry seedlings were obtained from seeds sown in January, 2012. The seedlings were initially peeled in cartridges with and then planted in pots (13 × 13 × 13 cm3) with peat substrate in May. The properties of the peat substrate were as follows: density 0.15 (g/cm3), total porosity 0.78 (m3/m3), water-holding 3 3 −1 −1 capacity 0.57 (m /m ), pH 5.6, P2O5 248 mg kg , and K2O 273 mg kg . From January to May and from November to the end of the experiment in May of the following year, the plants were grown in an unheated greenhouse. The greenhouse had a metallic frame with an arched roof, measuring 20 m long by 10 m wide, and a ceiling height of 3.5 m constructed in a north–south direction. The pots with plants were transferred and temporarily kept in open air from May to November. The resulting inflorescences were removed. Then, the pots were again transferred to an unheated greenhouse on tables with a height of 1.5 m. The pots were spaced 0.20 m apart. Plants started to bloom at the beginning of April in the following year. When grown outdoors and in the greenhouse, the plants received moisture and were fertilized according to the needs of the plants to ensure their normal growth at all times. The water deficit treatment started on 18 April and finished on 28 May. A randomized block experimental design was used in a 3 × 2 factorial arrangement, with three water treatments and two genotypes. The plants (seedlings) were arranged in three repetitions, each consisting of 12 plants, for a total of 36 plants evaluated for each variety and irrigation treatment. During the treatment, the applied water regimes were (1) full irrigation (I100), applying 100% of the estimated water crop requirements, and (2) 50% (I50) and (3) 25% (I25), applying half and a quarter of the full irrigation water supply, respectively. Under I100, 340 mL of water per potted plant was applied. This is the average amount of water that the substrate with plant can hold without excess drainage from the pot. This volume was determined during the previous 30 days before the irrigation treatment. During the same period, it was observed that watering was required every 2–4 days to maintain sufficient substrate moisture in pots with plants. This watering frequency was maintained in the WD study, in which the plants were watered a total of 15 times. The net irrigation for fully irrigated plants during our WD treatment was 5.1 L per plant (pot). In our experience, this amount of water corresponds to the volume that is normally consumed by growing strawberry seedlings for berry production in greenhouses in early spring (data not shown). For (I50) and (I25) plants, 170 mL (50%) and 85 mL (25%) were applied, respectively, at the same periodicity as that for I100 plants. From 22 April until the end of harvesting (28 May), ripe berries were picked, counted, weighed, and frozen at −70 ◦C to −80 ◦C every fourth day. The number of berries per plant, average weight, and yield per plant were measured. The standard deviation and correlation were established using the ANOVA method and Microsoft Excel. Plants 2020, 9, x FOR PEER REVIEW 9 of 11

previous harvests. Anthocyanins were extracted from frozen berries using 90% aqueous methanol, with 0.02% HCl at a 1:20 g:mL ratio [42], and concentrated in rotary evaporator at 37 °C. Residues were dissolved in acidified water (pH 2.4). The anthocyanin composi- tion was analyzed using an Agilent 1200 HPLC system with a DAD detector (Agilent, Germany); a reverse phase XBridge Shield RP18 (Waters, UK) analytical column (3.0 × 150 mm, particle size 3.5 μm) was used for separation. The mobile phase consisted of 10% acetic acid and 1% phosphoric acid (solvent A) and 100% acetonitrile (solvent B); HPLC gradient grade reagents were purchased from Sigma-Aldrich. The elution conditions were as follows: isocratic elution 0% B, 0–12 min; linear gradient from 0% B to 7% B, 12–15 min; to 45% B, 17 min; to 100% B, 20 min, and flow rate 0.7 mL min−1. All anthocyanins were Plants 2021, 10, 557 9 of 11 quantified at a detection wavelength of 520 nm using an external standard of cyanidin 3- O-glucoside (C3G), pelargonidin 3-O-glucoside (Pel3G), peonidin 3-O-glucoside (Pn3G), cyanidin 3-O-rutinoside (C3R), cyanidin 3-O-sambubioside (C3SAM), delphinidin 3-O- rutinosideThe air (D3R), temperature delphinidin was 3-O-glucoside highly variable (D3G), during peonidin the experiment: 3-O-rutinoside the (Peo3R), average and air temperaturemalvidin 3-O-glucoside varied from 11 (M3G).◦C to 24 All◦C, standa with ards significant were purchased temperature from increase Extrasynthese, after 8 May (FigureFrance,4 ).and Polyphenols Laboratories, Norway.

FigureFigure 4.4. AirAir temperaturetemperature outsideoutside andand insideinside thethe greenhouse.greenhouse.

AuthorThe Contributions: total and cumulative Conceptualization, concentrations R.R. and of V.K.; anthocyanin methodology, were T.S.; evaluated. software, TheV.B.; cumu-valida- lativetion, A.S., amount V.S., isand the V.B.; amount formal of analysis, anthocyanins V.B.; investigation, in a particular R.R.; harvest resources, plus V.S.; the data amount curation, in previousA.S.; writing—original harvests. Anthocyanins draft preparation, were R.R.; extracted writing—review from frozen and berries editing, using V.B.; 90% visualization, aqueous methanol,R.R.; supervision, with 0.02% V.S.; project HCl at administration, a 1:20 g:mL ratio V.K.; [ 42funding], and acquisition, concentrated V.K. in All rotary authors evaporator have read atand 37 agreed◦C. Residues to the published were dissolved version inof acidifiedthe manuscript. water (pH 2.4). The anthocyanin composition wasFunding: analyzed The research using an was Agilent funded 1200 by HPLCthe Research system Council with a of DAD Lithuania detector grant (Agilent, no. SVE-06/2012. Germany); a reverse phase XBridge Shield RP18 (Waters, UK) analytical column (3.0 × 150 mm2, par- ticleConflicts size 3.5of Interest:µm) was The used authors forseparation. declare no conflict The mobile of interest. phase consisted of 10% acetic acid and 1% phosphoric acid (solvent A) and 100% acetonitrile (solvent B); HPLC gradient References grade reagents were purchased from Sigma-Aldrich. The elution conditions were as fol- 1. Balasooriya, H.N.; Dassanayake,lows: K.B.; isocratic Senew elutioneera, S.; 0% Ajlouni, B, 0–12 S. Impact min; linear of elevated gradient carbon from dioxide 0% Band to 7%temperature B, 12–15 on min straw-; to berry polyphenols. J. Sci. Food45% Agric. B, 17 2019 min;, 99 to, 4659–4669, 100% B, doi:10.1002/jsfa.9706. 20 min, and flow rate 0.7 mL min−1. All anthocyanins were

2. Palencia, P.; Martínez, F.; Medina,quantified J.J.; Vázquez, at a detection E.; Flores, wavelength F.; López-Medina, of 520 J. nm Effects using of climate an external change standard on strawberry of cyanidin produc- tion. Acta Hort. 2009, 838, 51–54, doi:10.17660/ActaHortic.2009.838.6. 3-O-glucoside (C3G), pelargonidin 3-O-glucoside (Pel3G), peonidin 3-O-glucoside (Pn3G), 3. Bordonaba, J.G.; Terry, L.A. Manipulating the taste-related composition of strawberry fruits (Fragaria x ananassa) from different cyanidin 3-O-rutinoside (C3R), cyanidin 3-O-sambubioside (C3SAM), delphinidin 3-O- cultivars using deficit irrigation. Food Chem. 2010, 122, 1020–1026, doi:10.1016/j.foodchem.2010.03.060. 4. Casadesús, A.; Arabia, A.; rutinosidePujolriu, R.; (D3R), Munné-Bosch, delphinidin S. Differe 3-O-glucosidential accumulation (D3G), peonidinof tocochromanols 3-O-rutinoside in photosynthetic (Peo3R), and and non-photosynthetic tissues malvidinof strawberry 3-O-glucoside plants subjected (M3G). to reiterated All standards water weredeficit. purchased Plant Physiol. from Biochem. Extrasynthese, 2020, 155, 868–876, , doi:10.1016/j.plaphy.2020.06.038.and Polyphenols Laboratories, Norway. 5. Perin, E.C.; da Silva Messias, R.; Borowski, J.M.; Crizel, R.L.; Schott, I.B.; Carvalho, I.R.; Rombaldi, C.V.; Galli, V. ABA-depend- ent salt and drought stress improveAuthor Contributions: strawberry fruitConceptualization, quality. Food Chem.R.R. 2019 and, 271 V.K.;, 516–526, methodology, doi:10.1016/j.foodchem.2018.07.213. T.S.; software, V.B.; vali- 6. Antunes, A.C.; Acunha, T.d.S.;dation, Perin, A.S., E.C.; V.S., Rombaldi, and V.B.; formal C.V.; Galli, analysis, V.; V.B.;Chaves, investigation, F.C. Untargeted R.R.; resources, metabolomics V.S.; dataof strawberry curation, (Fragaria x ananassa ‘Camarosa’)A.S.; writing—original fruit from plants draftgrown preparation, under osmotic R.R.; stress writing—review conditions. J. and Sci. editing, Food Agric. V.B.; 2019 visualization,, 99, 6973–6980, R.R.; doi:10.1002/jsfa.9986. supervision, V.S.; project administration, V.K.; funding acquisition, V.K. All authors have read and 7. Weber, N.; Zupanc, V.; Jakopic,agreed J.; toVeberic, the published R.; Mikulic-Petkovsek, version of the manuscript. M.; Stampar, F. Influence of deficit irrigation on strawberry (Fragaria × ananassa Duch.) fruit quality. J. Sci. Food Agric. 2017, 97, 849–857, doi:10.1002/jsfa.7806. Funding: The research was funded by the Research Council of Lithuania grant no. SVE-06/2012. 8. Nora, L.; Dalmazo, G.O.; Nora, F.R.; Rombaldi, C.V. Controlled water stress to improve fruit and vegetable postharvest quality. In Water Stress; Rahman, I.M.M.,Conflicts Hasegawa, of Interest: H., Eds.;The authorsIntechOpen: declare Lo nondon, conflict UK, of2012; interest. Volume 25, pp. 59–72, doi:10.5772/30182.

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